Isolation and Structural Studies of the Monocyte Locomotion Inhibitory Factor (MLIF) Produced by Entamoeba histolytica

Abstract

The supernatant fluid of axenically grown E. histolytica HM-1:IMSS (and virgin axenic medium as control) was obtained by centrifugation and ultrafiltration, and applied to gel-sieve (Sephadex G15) chromatography (1). The 478– 735-Da chromatographic fraction was subjected to reverse phase HPLC-C18. Only the second of five closely emerging peaks revealed MLIF activity (absent from the virgin axenic medium) and was, in turn, subjected to mass-spectrometry: MALDI-MS (2) and tandem MS/MS (TSQ-LC-MS) (3), and Edman sequencing (4). Once the primary chemical structure was known (5), a 96% pure custom MLIF (American Peptide Corp., Sunnyvale, CA, USA) was tested in vitro (human monocyte locomotion in Boyden chambers, as well as monocyte and neutrophil respiratory burst by chemiluminescence) and in vivo (delayed hypersensitivity skin reactions to 1-chloro-2-4 dinitrobenzene in guinea pigs) alongside the native MLIF.